Streptococcus pneumoniae is a significant cause of morbidity and mortality in pediatric, geriatric, and immunocompromised populations. Until the mid-sixties, this pathogen was uniformly susceptible to penicillin, but since then the incidence of resistance to this antibiotic (as well as many others) has increased to such an extent that infections due to penicillin-resistant pneumococci have become a major threat in many countries. With respect to the USA, a nationwide survey showed that 5% of the strains isolated in hospitals during the period 1979-'87 were intermediately resistant to penicillin (minimum inhibitory concentration (MIC) = 0.1-1 mg/L), and that 0.02% were highly resistant (MIC>1 mg/L). Under the aegis of a Children's Hospital (Washington, D.C.) - NIDR clinical research training program, a study was initiated to assess the prevalence of resistance to penicillin, as well as several other antibiotics, among pneumococci isolated from patients attending this hospital. This June 1992-May '93 survey showed that 8.3% of the 108 isolates were intermediately resistant to penicillin, and that 4.6% were highly resistant. Moreover, at least 40% of the penicillin-resistant strains were also resistant to frequently used oral and parenteral cephalosporins and carbapenems. Also of great concern was the finding that all isolates were resistant to trimethoprim/sulfamethoxazole (Pikis, A., Akram, S., Donkersloot, J.A., Campos, J.M. and Rodriguez, W.J. Arch. Pediatr. Adolesc. Med. 149:30-35, 1995). As a result of these findings, a study to define, at the sequence level, the molecular epidemiology and mechanism of trimethoprim resistance in S. pneumoniae was initiated. To this end, the trimethoprim-resistance determinant of several clinical isolates will be cloned, sequenced, and analyzed. The identification of specific residues that might be responsible for resistance will also require the cloning and sequencing of the trimethoprim target protein from one or more sensitive strains. Initially, the PCR, with primers based on sequences that are conserved in the resistant isolates, will be tried for cloning. Alternatively, a gene library of the sensitive strain can be prepared and screened with gene probes based on resistant isolates. The comparative sequence analysis is expected to reveal the molecular mechanism of trimethoprim resistance in the Washington DC area. This information will be combined with modeling of the trimethoprim-target protein to design novel antibiotics effective against trimethoprim-resistant streptococci.